Generally speaking, plant transformation is performed using a technique of introducing foreign DNA into plant cells, which allows the foreign gene to be retained/expressed in progenies of fertile seeds or vegetative propagated plants. Quite a few monocotyledons and dicotyledons were actually transformed using the technique. Not only were crops of maize, rice, wheat, barley, sorghum, soybean, rapeseed, sunflower, cotton, potato, tomato produced by plant transformation, but those of fruits and other vegetables were also produced, and a strong interest exists for the commercial use of the technique.
Publicly known plant transformation methods include physical/chemical methods such as polyethylene glycol, electroporation, particle gun, etc. (direct introduction of DNA) and biological methods using the function of the Agrobacterium bacterium (indirect introduction of DNA). The direct introduction of DNA is often associated with problems such as fragmentation of the target gene during introduction, and an introduction of a high number of copies. As a result, there is a frequent occurrence of transformed plants that do not express the target gene or transformed plants that show weak, abnormal expression (gene silencing). A use of protoplasts in a method prolongs the culture period, which tends to induce non-fertile seeds or malformation in the obtained transformants as a result of mutations during culture. In contrast, such problems are unlikely to occur when the gene is introduced by the Agrobacterium bacterium. In the method using Agrobacterium, the target gene is introduced via regulating expression of the gene group of the virulent region of the Ti or Ri plasmid (vir region). The target gene is introduced into the plant by the work of the protein group encoded in the vir gene, going through many processes including recognizing the interaction and signaling between plant cells and bacterium, inducing expression of the vir gene, creating a Type IV secretion route, recognizing the T-DNA border repeat sequences, creating the T-DNA strand, and transferring the T-DNA strand to the plant cell and then to the nucleus, and integrating the T-DNA into the plant nucleus genome. Such process restrains the number of introduced copies of the target gene and prevents the gene from being fragmented in introduction. As a result, it was possible to prolifically and stably produce transformed plants exhibiting a high expression of the target gene, so that the variation in the population of transformation products is lower than the direct introduction of DNA.
As seen, Agrobacterium method is quite suitable for plant transformation, but actually, the success and efficiency of plant transformation vary greatly according to the plant species, the genotypes and the plant tissue types that are used (Potrykus et al., 1997). There are still many plants that cannot be transformed with sufficient efficiency, and at the current stage, the crops for which a large number of transformed plants can be provided are limited. There is thus a strong need for improved methods to solve such problem.
Many vectors for transformation have been developed to date in an effort to solve the above problems. The background of vector development is explained below. Conventionally, the large size of the Ti plasmid of a wild-type Agrobacterium at 190 kb or longer made it difficult to use the standard gene engineering method to insert genes into the T-DNA on the plasmid. Hence, methods were developed to insert foreign genes onto the T-DNA. The following bacterial strains having disarmed Ti plasmids, which are tumor-inducing Ti plasmids whose plant growth regulator synthetic gene is deleted from T-DNA, were first constructed: LBA4404 (Hoekema et al., 1983), GV3850 (Zambryski et al., 1983), GV3TillSE (Fraley et al., 1985), C58-Z707 (Hepburn et al., 1985), GV3101::pMP90 (Koncz and Schell, 1986), GV3101::pMP90RK (Koncz and Schell, 1986), GV2260 (McBride and Summerfelt, 1990), NTI (pKPSF2) (Palanichelvam et al., 2000).
These bacterial strains were used to develop two methods for introducing the desired gene into the T-DNA of a Ti plasmid of Agrobacterium, or introducing the other plasmid having the T-DNA with the desired gene into Agrobacterium. One of these methods is called an intermediate vector method (Fraley et al., 1983), which allows easy genetic manipulation and insertion of the desired gene, and this method is performed by introducing intermediate vectors that can be replicated by E. coli into the T-DNA of disarmed Ti plasmids of Agrobacterium by homologous recombination through triparental mating (Ditta et al., 1980). Another method is called the binary vector method, which is based on the finding that the vir region is required for integration of T-DNA into plants but it does not have to be located on the same plasmid as T-DNA to perforin its function (Hoekema et al., 1983) (Lee and Gelvin, 2008). This vir region includes virA, virB, virC, virD, virE and virG. The tern “binary vector” refers to a vector carrying T-DNA integrated into a small plasmid replicable in both Agrobacteriumand E. coli, and this binary vector is introduced into Agrobacterium having a disarmed Ti plasmid before use. Introduction of a binary vector into Agrobacterium can be accomplished in any known manner, for example, by electroporation or triparental mating. Examples of a binary vector include pBIN19 (Bevan, 1984), pBI121 (Jefferson, 1987), pGA482 (An et al., 1988), etc., and many new binary vectors have been constructed based on these vectors and used for transformation (Lee and Gelvin, 2008).
Agrobacterium A281 (Watson et al., 1975) is a super-virulent bacterial strain whose host range is wide, and its transformation efficiency is higher than other strains (Komari et al., 1986). This feature is based on pTiBo542 of Ti plasmid having A281 (Jin et al., 1987).
Three transformation systems that use pTiBo542 have been developed. The first uses bacterial strains that have a disarmed Ti plasmid of pTiBo542, namely, EHA101 (Hood et al., 1986), EHA105 (Hood et al., 1993), AGL0 (Lazo et al., 1991) or AGL1 (Lazo et al., 1991). This system is used as a system with a high transformation ability to transform various plants, and such ability is obtained by applying these strains to the above binary vector system.
The second is a super-binary vector system (Hiei et al., 1994; Ishida et al., 1996). This system is a kind of a binary vector system because it consists of a disarmed Ti plasmid having the vir regions (all of virA, virB, virC, virD, virE and virG, virJ) and a plasmid with T-DNA. However, it differs in that it uses a super-binary vector wherein a 14.8 kb KpnI fragment taken from the pTiBo542 vir region (a part of the virD1 gene, the virB gene, the virC gene and the virG gene) has been introduced into the plasmid with T-DNA, i.e., the binary vector. The KpnI fragment was described to be 15.8 kb in the first article (Jin et al., 1987), but it is 14.8 kb to be correct. Besides, homologous recombination via triparental mating can be used as a convenient technique to introduce the T-DNA region carrying a desired gene into Agrobacterium containing a super-binary vector (Komari, 1996). It has now been clarified that the super binary vector system would provide an extremely high transformation efficiency in various plant species (Saito et al., 1992; Hiei et al., 1994; Ishida et al., 1996). In particular, it is reported that the super binary system shows an excellent effect for maize transformation (Ishida et al., 1996). In addition, Khanna and Daggard (2003) was successful in obtaining a wheat transformat that was not obtainable from a combination of LBA4404 and a normal binary vector pHK22 by using a combination of an Agrobacterium strain LBA4404 that is not super-virulent and a super binary vector pHK21.
The third is a system in which plasmid pTOK47 (Jin et al., 1987), which is a plasmid carrying a 14.8 kb KpnI fragment (a part of virD1, virB, virC and virG) cut out from the vir region of pTiBo542, is additionally introduced to the binary vector system as a booster vector for improving transformation efficiency. A binary vector system that further includes a booster vector is generally called a “ternary vector system,” but the booster vector comprises a 14.8 kb KpnI fragment as mentioned above, so this vector is particularly referred to as the “super-ternary vector system” in the present specification. In this system, plasmids, which can coexist in Agrobacterium and have different origins of replication (ori) belonging to different incompatibility groups, are used. Each ori needs a coding region of the Rep protein (initiator), and they are normally used in close proximity. It has been reported that the super ternary vector system using pTOK47 shows a high transformation efficiency in many plant species (Wenck et al., 1999; Tang, 2003; Dong and Qu, 2005; Arokiaraj et al., 2009). The origin of replication (ori) of pTOK47 belongs to the IncW incompatibility group, and the plasmids having T-DNA in the reports of these ternary-vector systems use mainly an ori belonging to the IncP incompatibility group. The super ternary vector system using the booster vector pTOK47 is reported as being useful for the transformation of maize, although somewhat less than the super binary vector system (WO 2007/148819 A1). There is also a report of wheat transformation by using a super ternary vector system, in which a 14.8 kb KpnI fragment derived from the vir region of pTiBo542, similar to pTOK47, is located on a plasmid other than the plasmid with the T-DNA (Amoah et al., 2001; Wu et al., 2008). The vector system used by Amoah et al. (2001) and Wu et al. (2008) consists of pGreen which contains only the IncW ori but lacks the rep gene (Hellens et al., 2000), and pSoup which contains the rep gene of IncW in the trans portion and also contains the oriV of IncP that is necessary for self replication (Hellens et al., 2000). That is, the plasmids pSoup and pGreen are stably maintained in the Agrobacterium by pSoup supplementing the replication of pGreen. This system is thus specifically called the dual binary vector system. However, it is actually recognized to be a super ternary vector system consisting of three types of plasmids, the disarmed Ti plasmid, the binary vector pSoup and the ternary vector pGreen.
Knowledge Relating to the Vir Region of pTiBo542
Jin et al. (1987) assessed the crown gall formation ability by retaining super-vir (the vir region of pTiBo542) of various lengths as additional matters in Agrobacterium. In the test, the super-virulent strain A281 containing the entire super-vir region was used with the normal strain A348. The result showed that an addition of virB and virG of super-vir is important to improve transformation ability (crown gall formation ability) in both strains. On the other hand, no effect was observed for addition of the virD or virE region (Jin, S., Komari, T., Gordon, M. P., and Nester, E. W. (1987). Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J. Bacteriol. 169, 4417-4425. Table 3 and FIG. 3).
In addition, Hiei et al (1994) used the disarmed strain EHA101 (pIG121Hm) of the super-virulent strain A281 and a super binary vector LBA4404 (pTOK233) having normal virulence (entire normal vir region) but also retaining a part of the super-vir (virB, virC, virG) on the binary vector to compare their transformation ability concerning rice. As a result, the latter was verified as having a higher transformation efficiency. This suggests that a use of a partial region (virB and virG) provides a higher transformation ability than a use of the entire region when using the transformation vector of the super-vir region.
Concerning virD, virD1 and virD2 cut the border sequence and produce T-DNA. In particular, virD2 forms a complex with T-DNA. virD3 has a low conservative property, and is considered to be unnecessary for the transfer of T-DNA. virD4 constitutes a type IV secretion system together with the virB gene group. virD5 is considered to be an accessory protein having a signal function of type IV secretion system (Ream, W. (2008). Production of a mobile T-DNA by Agrobacterium tumefaciens. In Agrobacterium, T. Tzfira and V. Citovsky, eds (New York: Springer Science+Business Media, LLC), pp. 280-313.)
Generally speaking, a small vector is more preferable, since incorporation of a desired DNA is easier in a smaller vector. It is also presumed that the foreign DNA that can be stably maintained in E. coli is 1200 to 1500 kb per cell (Tao, Q., and Zhang, H.-B. (1998). Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors. Nucleic Acids Research 26 4901-4909). As such, E. coli cannot stably maintain a plasmid that is larger than a certain size. For example, there are 30 to 40 copies of plasmid having a ColE1-derived origin of replication per an E. coli cell, so the E. coli cell cannot maintain plasmids that are larger than 30 to 40 kb.
There was no knowledge concerning the vir region of pTiBo542 suggesting that the virD region and the vin region are particularly useful for transformation using the Agrobacterium bacterium.